Optical Apparatus

ABSTRACT

An optical apparatus for illuminating particles and for detecting emissions from illuminated particles in a flow cytometer having an optical configuration aligned with a flow or particles.

This application is a divisional application of, and claims benefit ofand priority to, U.S. patent application Ser. No. 10/990,648, filed Nov.16, 2004, issuing May 22, 2007 as U.S. Pat. No. 7,221,453, which itselfis a continuation application of, and claims the benefit and priorityof, U.S. patent application Ser. No. 09/355,461, filed Jul. 29, 1999,issuing Nov. 16, 2004 as U.S. Pat. No. 6,819,411, which itself was aUnited States National Phase patent application of InternationalApplication No. PCT/NZ98/00009, published Aug. 6, 1998, filed Feb. 2,1998, which itself claims the benefit of and priority of New ZealandProvisional Specification Number 314169 filed Jan. 31, 1997, eachabove-mentioned application hereby incorporated by reference.

BACKGROUND OF THE INVENTIVE TECHNOLOGY

This invention relates to an optical apparatus. In particular, althoughnot exclusively, this invention has application to the field of flowcytometry. However, it is to be understood that several of the inventiveaspects have application beyond flow cytometry and may have broadapplication in the field of optics generally. For example, severalaspects of the invention may be used in photometry or optical particledetection apparatus.

Generally when illuminating a particle or an object for analysis, thelight source is directed onto the particle from a single direction. Ananalysis may be made of light reflected or produced by the particle e.g.fluorescence to reveal certain properties of the particle. Theparticular portion of the particle illuminated depends on theorientation of the particle with respect to the light source. Where theparticle or object is asymmetrical, the light measurements will varydepending on which portion is illuminated, making it difficult toanalyze the particle or object as a whole.

Such difficulties are encountered in flow cytometry since it is commonfor particles being analyzed to be asymmetrical e.g. mammalianspermatozoa.

Flow cytometers are often used to measure the properties of cells orparticles which are carried in a stream of fluid. The stream isgenerally comprised of a sheath fluid into the centre of which isinjected a narrow aqueous suspension of cells/particles. The sheathfluid focuses the sample cells/particles into single file. The streamcontaining the particles/cells passes through an inspection point whichis the focus of an intense light beam. The particles/cells may have beenstained with a light-sensitive stain which when illuminated, will absorbthe incident light and fluoresce. Light scatters off the particlesand/or alternatively causes fluorescence. This scattered or fluorescentlight is then measured by a detector generally aligned with the incidentbeam. The characteristics of the detected signal(s) such as peakintensity, peak area or other characteristics of interest may then beused to derive properties of the particle, for example size.

In a flow cytometer with sorting capability (as opposed to a purelyanalytical instrument) the detected signal(s) may be used to triggersorting hardware which can be programmed to divert droplets from thestream of fluid. The sorting criteria will vary with the application,for example, the sorting may be conducted according to size or, in thecase of spermatozoa, the DNA content of the cell.

One problem with conventional flow cytometers is that particle asymmetryoften renders the optical characteristics of a particle difficult tomeasure. For example, a flat particle can pass through the inspectionpoint with a random orientation. Thus, the intensity of the resultantscattered or fluorescent light may vary according to particleorientation and the detectors will measure different light intensitiesat different locations.

Thus, particle asymmetry can lead to a reduced resolution of measurementof the particles. It follows that, in cytometers with a sortingcapability, this reduced resolution in measurement of the particlesresults in a decreased ability to accurately separate populations ofcells with different optical properties. Such a problem is encounteredin separation of male and female mammalian sperm.

In mammals, sperm carry the sex determining chromosomes and the totalDNA content found in male and female sperm may differ. For example, incattle the difference in the DNA content between male and female spermis approximately 4%. This difference in DNA provides a means by whichsperm may be separated in a sorting flow cytometer, making apredetermination of an offspring's sex possible when artificial breedingof animals is carried out. Utilizing such a technique in artificialbreeding would offer considerable economic advantages in livestockmanagement, but is currently made difficult by the asymmetric geometryof the flat sperm head. As an example, bull sperm are flat cells withhead dimensions of approximately 10 microns by 4 microns by 1 micronattached to a 40 micron flagellum. The asymmetric properties of the bullsperm head result in a high variation in both scattered light andfluorescent light emission with sperm orientation. In particular,fluorescent emission varies by a factor of two with sperm orientation(see DNA Contention Measurements of Mammalian Sperm. CYTOMETRY 3:1-9[1982]), effectively masking the 4% variation in intensity due to thesex of the sperm.

A number of flow cytometric systems have been developed in an attempt toovercome the problems encountered when analyzing asymmetric particlessuch as sperm cells.

One flow cytometric system that has been developed in an attempt toovercome this problem introduces asymmetric cells traveling in a slowmoving stream into the middle of a fast flowing sheath stream.Hydrodynamics then tends to align the asymmetric cells with their longaxis parallel to the direction of the fast flowing sheath stream.

While this approach tends to reduce the vertical variation of lightintensity from asymmetric particles, the radial variation remains. Thissystem has been further refined so as to further reduce theorientation-related variation in the detected light intensity ofparticles.

The system developed by Pinkel et al. (see Flow Cytometry in MammalianSperm. Progress Morphology and DNA Measurement. THE JOURNAL OFHISTOCHEMISTRY AND CYTOCHEMISTRY 24:353-358 [1979]), showed that theorientation of bull sperm could be further aligned by bevelling the endof the tube which injected the sample stream (i.e. that which containsthe sperm) into the sheath flow.

The system which attempted to overcome the problems of flow cytometricanalysis of asymmetric cells was that described by Johnson (see SexPreselection by Flow Cytometric Separation of X AND Y Chromosome BearingSperm Based on DNA Difference: A review. REPRODUCTIVE FERTILITYDEVELOPMENTS 7:893-903[1995]), in relation to separation of bull spermby sex. Johnson's approach utilized two detectors; one in line with theilluminating laser beam (the 0 degree detector) and one at right anglesto the beam (the 90 degree detector). Sperm emit fluorescencepreferentially through their narrow edges. Johnson determined whichsperm were aligned edge-on to the 90 degree detector by detecting thebright emission from their edges, and used the 0 degree detector formeasuring the flat-face emission from only the aligned sperm.

However, this system still had a number of drawbacks. One drawback wasthat it was a requirement for this system that the sample flow be movingslowly with respect to the sheath flow, thereby reducing samplethroughput. A further drawback was that it only produces good alignmentat very low flow rates. At the optimal flow rate, which produced thegreatest number of aligned cells per second, only 40% of cells werealigned. Thus, the number of aligned cells had been increased from 10%to 40%, but approximately 60% of the cells remained unaligned, andfurther, due to the requirement of a low flow rate, there was areduction in system throughput.

It will be appreciated that the rejection of unaligned cells againreduces the processing rate of this system and unnecessarily wastessperm cells.

One system which moved towards radial light collection was theEllipsoidal Collector described by Skogen-Hagenson et al (see A HighEfficiency Flow Cytometer, CYTOCHEMISTRY 25:784-789[1977]), whodeveloped a light collection system based on a hollow “egg shaped” brassreflector. The reflector surface was elliptical in cross-section andlight from the inspection point at one focus was collected at the secondfocus. This system was demonstrated to have an ability to reduce theorientation dependence observed with bull sperm.

However, it still had orientation dependent illumination, (i.e. Lightsource coming from a single direction). A further problem with thissystem is that it is unable to provide a particle sort function (i.e.according to sperm sex).

A further system which implemented both symmetric illumination andsymmetric light collection was the Epi-Illumination system described byGarner et al (see Quantification of the X and Y Chromosome BearingSpermatozoa of Domestic Animals by Flow Cytometry, BIOLOGY OFREPRODUCTION 28:312-321[1983]).

In this system the sample stream traveled directly towards a highnumerical index microscope objective lens and was diverted sidewaysafter the stream had passed through the focal point of the lens.Illumination was delivered through the lens and light was collected backthrough the lens.

While this system also demonstrated a good ability to eliminate theorientation dependencies of bull sperm, it was also incapable ofmodification for high speed sorting. This was due to its sidewaysdiversion of the sperm immediately after passing through the focalpoint.

Earlier systems have also relied on laser light, because of theintensity of laser light sources. Unfortunately, such laser systems canbe quite expensive and only add to the cost of devices such as flowcytometers. Because lasers typically deliver a single wavelength oflight, use of lasers also has made it difficult to utilize a singlelight source to provide a variety of wavelengths of light, e.g. inconjunction with filters that filter out all but the desiredwavelengths.

Furthermore, previous systems have often required the precise alignmentof optics in order to accomplish a proper delivery of electromagneticradiation onto the cell under analyzation or collection of fluorescenceemitted by a cell. This can be a tedious process that adds to theexpense of the analyzation instruments. Hence, there is a need for asystem, e.g., in flow cytometry, in which the optics that focus andcollect electromagnetic radiation for measurement purposes are quicklyand easily established in their proper orientation.

It is an object of the present invention to overcome the afore mentionedshortcomings of known optical apparatus with particular application toflow cytometers. It is also an object of the invention to provide thepublic with a useful choice.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention there isprovided an optical apparatus including: a prism having a conicalportion with an apex at a forward end of the prism and a central axisextending through the apex of the prism; an optical arrangementincluding a source of electromagnetic radiation, the optical arrangementadapted to direct an incident beam of electromagnetic radiation onto theapex of the conical portion in a direction substantially aligned withthe central axis of the conical portion; and a reflective surfaceprovided behind the apex of the prism; such that the beam refracted bythe prism will be reflected by the reflective surface back through theprism to project from the forward end of the prism as an annular beam ofelectromagnetic radiation.

The optical apparatus described above thereby serves to produce anannular beam of electromagnetic radiation from a single beam ofelectromagnetic radiation incident onto the apex of the conical portion.Preferably, the arrangement is such to provide the beam with a constantcross section to produce a cylindrical tube of light. The prism may alsoinclude a cylindrical base portion at a rear end thereof which has acircular cross section corresponding to the cross section of the base ofthe conical portion.

In accordance with a second aspect of the present invention there isprovided an optical apparatus including: a prism having a pyramidalportion with an even number of inclined faces meeting at an apex at aforward end of the prism and a central axis extending through the apexan optical arrangement including a source of electromagnetic radiation,the optical arrangement adapted to direct an incident beam ofelectromagnetic radiation onto the apex of the pyramidal portion in adirection substantially aligned with the central axis of the pyramidalportion; and a reflective surface provided behind the apex of the prism;such that the beam refracted by the prism will be reflected by thereflective surface back through the prism to project from the forwardend of the prism as a number of parallel beams.

It is required that the pyramidal portion have an even number ofinclined faces since the optical geometry is such that the beams crossthe prism to reflect from the opposing face. Apart from this constraint,the number of the inclined faces is not limited. For example, there maybe 4, 6, 8 . . . 12 inclined triangular faces converging towards theapex of the pyramidal portion. Preferably, the pyramidal portion alsoincludes a base portion with a cross section corresponding to the baseof the pyramidal portion. For example, where the pyramid has fourinclined faces an appropriate base portion would be a rectangular prismor a cube.

In either of the first two aspects of the invention, the reflectivesurface may be provided at the rear end of the prism. However, theinvention is not limited to this arrangement and may potentially bedisposed within the prism itself. Another preferred arrangement is forthe reflective surface to be spaced from the base portion. Anotherdesirable feature is that this spacing be adjustable to provide avariable annular beam diameter.

However, where the reflective surface is spaced from the prism theelectromagnetic radiation may suffer losses from multiple interfacereflection. However, as such a design would have a reduced length fromthe front to the rear end, the transmission losses would be less thanfor a longer prism with the reflective surface provided at the rear end.

Suitably the prisms are manufactured from optical glass such as BK7optical glass. However, where the application is intended for use withUV electromagnetic radiation, it is preferred to manufacture the prismfrom UV-suitable material such as fused silica. In such an application,it is also desirable that the reflective surface be comprised of aUV-grade mirror to increase the transmission efficiency of the opticalapparatus.

As mentioned above, the optical apparatus may be used with ultra-violetradiation, preferably produced from a laser source. The electromagneticradiation may also include other wavelengths including those in thevisible spectrum. Suitably, the incident electromagnetic radiation is inthe form of a collimated beam.

The optical apparatus described above in connection with the first twoaspects may desirably be used in combination with a paraboloid reflectorhaving an internal paraboloidal-shaped reflective surface and an opticalaxis. Such a reflector will be oriented to receive, on its reflectivesurface, the electromagnetic radiation projected from the forward end ofthe prism. It will be appreciated that such a paraboloidal-shapedreflective surface will have a focus at which all light parallel to theoptical axis and incident onto the reflective surface will be directed.In other words, the parallel electromagnetic radiation projected fromthe prism will be received onto the paraboloid reflector to converge atthe focus. Such a concentration of electromagnetic radiation may havemany useful and varied applications in the field of optics. Inparticular, the invention is capable of providing radially symmetricillumination to the focus of the paraboloid reflector. The term“radially symmetric” means that for every beam of incident radiation tothe focus, a substantially diametrically opposite beam will be incidentto the focus. Each beam of the radially symmetric illumination may havethe same angle to the optical axis of the paraboloid reflector. Thus aconvergent disc of electromagnetic radiation onto the focus will beincluded in the definition of “radially symmetric”. Such a convergentdisc can be achieved through the use of the first-described opticalapparatus in combination with the paraboloid reflector. Any object canbe placed at the focus of the paraboloid reflector for illumination andinspection. As will be discussed with following aspects of theinvention, the apparatus has particular application to flow cytometry inthat a flow source may be provided to direct particles through the focusof the paraboloid reflector.

It will be understood that the source of electromagnetic radiation maynot be directed directly at the apex of the prism and the inventionallows for the use of mirrors and other reflectors as desired. Inparticular, a second reflector may be disposed between the prism and theparaboloid reflector, the second reflector having reflective portions toreflect the incident beam from the source onto the apex of the prism andtransmitting portions to transmit the beam(s) projected from the forwardend of the prism.

However, the invention is not limited to the particular prisms describedin the forgoing aspects of the invention. Other optical configurationsare envisaged to produce the projected annular beam or parallel beams ofelectromagnetic radiation. Furthermore, other types of reflectors whichfocus incident radiation towards one or more foci could be adopted.

Accordingly, a third aspect of the invention provides an opticalapparatus including an optical configuration adapted to produce anannular beam of electromagnetic radiation having a central axis orplurality of beams of electromagnetic radiation wherein said pluralityof beams are evenly spaced from a central axis; and a focusing reflectorhaving an internal reflective surface having an optical axis and one ormore foci, the reflector being oriented to receive, onto its reflectivesurface, the annular beam or the plurality of beams of electromagneticradiation.

For example, the optical element may comprise any known reflectiveaxicons as well as the particular prisms described above which, in somecases are also axicons. For example, the axicon may comprise an innerconical mirror with forward reflective surfaces surrounded by an outerconical mirror with forward reflective surfaces wherein the optical axesof the two mirrors are aligned. The reflective surfaces form the letter“W”, hence the name w-axicon or waxicon.

Preferably, the focusing reflector has an internal reflective surfacewhich is paraboloid in shape. The use of the term “paraboloid reflector”used throughout the specification and the claims will be understood tomean “a reflector conforming to the shape of a paraboloid ofrevolution”. The term is also to be understood to mean “a portion of afull paraboloid of revolution”. Similarly, in regard to the optical axisof a paraboloid, such an axis may also be considered to be the parabolicor central axis of the paraboloid.

As mentioned in connection with the foregoing aspect of the invention,the apparatus may be incorporated into a flow cytometer including a flowsource to produce a flow of particles to be analyzed in which the flowsource is adapted to direct the flow of particles substantially throughone of the foci of the reflective surface. Suitably the flow source canbe adapted to substantially align the flow with the optical axis of thereflective surface. Moreover, an aperture may be provided in thefocusing reflector for passage of the flow therebeyond.

It is desirable that the present invention will be used in a flowcytometer accommodating a sorting function. Thus, the flow means mayinclude a nozzle and the flow cytometer may incorporate electrostaticdroplet deflection sorting apparatus below the aperture in the focusingreflector.

In accordance with a fourth aspect of the present invention there isprovided an optical method including: providing a prism having a conicalportion with an apex at the forward end, a central axis extendingthrough the apex and a reflective surface provided behind the apex ofthe prism; directing an incident beam of electromagnetic radiation ontothe apex of the conical portion in a direction substantially alignedwith the central axis of the conical portion to produce an annular beamof electromagnetic radiation projecting from the forward end of theprism.

In accordance with a fifth aspect of the present invention there isprovided an optical method including: providing a prism having apyramidal portion with an even number of inclined faces meeting at anapex at a forward end of the prism, a central axis extending through theapex and a reflective surface provided behind the apex of the prism;directing an incident beam of electromagnetic radiation onto the apex ofthe pyramidal portion in a direction substantially aligned with thecentral axis of the pyramidal portion to produce parallel beams ofelectromagnetic radiation projecting from the forward end of the prism.

In accordance with another aspect of the present invention there isprovided an analyzation instrument including: a flow source to produce aflow of particles to be analyzed, the flow source being adapted todirect the flow of particles through an inspection zone; an opticalarrangement including a source of electromagnetic radiation, the opticalarrangement adapted to converge substantially coplanar, substantiallyradially symmetric electromagnetic radiation towards the inspectionzone.

Preferably, the electromagnetic radiation coverages in the form of adisc disposed symmetrically relative to the central axis.

In accordance with yet another aspect of the present invention there isprovided a method of analyzing including: providing a flow of particlesto be analyzed; directing the flow of particles to be analyzed throughan inspection zone; converging substantially coplanar, substantiallyradially symmetric electromagnetic radiation towards the inspectionzone.

In accordance with a further aspect of the present invention there isprovided an analyzation instrument including: a flow source to produce aflow of particles to be analyzed; a source of electromagnetic radiation;a reflector adapted to reflect at least a portion of the electromagneticradiation at the flow of particles to illuminate the flow of particles;an optical configuration including a sensor adapted to senseelectromagnetic radiation; wherein the reflector is also adapted toreflect, to the optical configuration, any electromagnetic radiationproduced as a result of the illumination of the flow of particles.

Thus the reflector described in accordance with this aspect serves thedual purpose of reflecting the electromagnetic radiation onto the flowof particles as well as collecting the electromagnetic radiation fortransmission to the sensor. Such a configuration can be achieved withthe use of a reflector having an internal reflective surface which isparaboloid in shape. It will be understood that any use of the term“illumination” or “illuminate” is not restricted to merely visibleillumination as non-visible wavelengths may also be used. As mentionedpreviously, in certain applications ultra violet radiation may be used.Furthermore, reference to electromagnetic radiation “produced” by theparticle may include any florescence produced by the particles as aresult of the incident illumination and/or any light scattered by theparticles. It should also be understood that “irradiate” is intended tohave the same meaning as “illuminate”.

In accordance with a still further aspect of the present invention thereis provided a method of analyzing including providing: a flow ofparticles to be analyzed; providing a source of electromagneticradiation; reflecting with a reflector at least a portion of theelectromagnetic radiation to illuminate the flow of particles;reflecting with the reflector at least a portion of any electromagneticradiation produced from the illumination of the flow of particles;sensing a portion of the electromagnetic radiation produced from theillumination of the flow of particles.

In accordance with still a further aspect of the present invention thereis provided a flow cytometer including: a flow source to produce alinear flow of particles to be analyzed, the flow source being adaptedto direct the flow of particles through an inspection zone; an opticalarrangement adapted to converge electromagnetic radiation onto the flowat the inspection zone in a radially symmetric manner about theinspection zone; a collector to collect electromagnetic radiation eitherproduced or deflected from the particles in the flow; a processor toderive, from the collected electromagnetic radiation, predeterminedinformation relating to each of at least some of the particles in theflow; and a correlator to correlate the derived information with theassociated particle downstream of the inspection zone.

As mentioned previously, the radially symmetric illumination may beprovided in the form of a continuous disc convergent towards theinspection zone. Another preferred radially symmetric arrangement of theillumination is in the form of discreet beams converging towards theinspection zone. Either way, the particle is illuminated evenly from allsides.

In accordance with a further aspect of the present invention there isprovided a flow cytometer including: a flow source to produce a linearflow of particles to be analyzed, the flow source being adapted todirect the flow of particles through an inspection zone; and an opticalarrangement including a focusing reflector having an internal reflectivesurface with one or more foci, the optical arrangement adapted toconverge electromagnetic radiation onto the flow of particles at theinspection zone by reflection from the focusing reflector, the focusingreflector being oriented such that one of the one or more foci issubstantially coincident with or located within the inspection zone.

Various embodiments of the focusing reflector have been envisaged. Inone such embodiment the focusing reflector comprises a paraboloidreflector having an internal reflective surface of paraboloidal-shape.The flow of particles will thus flow through the focus of the paraboloidreflector at which the electromagnetic radiation is converged. Inanother embodiment of the invention the focusing reflector may have anellipsoidal reflective surface with two foci and an optical axisextending between the two foci. In particularly preferred versions ofthis, the flow source is oriented so that the flow of particles isaligned with the optical axis of the reflective surface. Moreover, anyforms of the focusing reflector may be provided with an aperture for thepassage of flow beyond the focusing reflector. Such an embodiment isparticularly adapted for use in a sorting flow cytometer which collectsthe electromagnetic radiation produced from the particles in the flow,processes the collected electromagnetic radiation to derivepredetermined information relating to each of at least some of theparticles in the flow and correlates the derived information with theassociated particle downstream of the inspection zone. In this way, thesorting flow cytometer can not only analyze the particles in the flowbut sort the particles according to predetermined sets of selectioncriteria. A preferred type of sorting flow cytometer is a jet-in-airflow cytometer.

In another aspect of the present invention there is provided a flowcytometer including: a flow source to produce a flow of particles to beanalyzed, the flow source being adapted to direct the flow of particlesthrough an inspection zone; an optical arrangement including a source ofelectromagnetic radiation, the optical arrangement adapted to directelectromagnetic radiation onto the flow of particles, at the inspectionzone; a collector to collect electromagnetic radiation either producedor deflected from the particles, the collector having an internalreflective surface with an optical axis and one or more foci, whereinthe collector is oriented such that the flow of particles issubstantially aligned with the optical axis.

In yet another aspect of the present invention there is provided a flowcytometer including: a flow source to produce a flow of particles to beanalyzed, the flow source being adapted to direct the flow of particlesthrough an inspection zone; an optical arrangement including a source ofelectromagnetic radiation, the optical arrangement adapted to directelectromagnetic radiation onto the flow of particles, at the inspectionzone; a collector to collect electromagnetic radiation either producedor deflected from the particles, the collector having an internalreflective surface with an optical axis and one or more foci, whereinthe collector is disposed such that one of the one or more foci issubstantially coincident or located within the inspection zone; aprocessor to derive, from the collected electromagnetic radiation,predetermined information relating to each of at least some of theparticles in the flow; and a correlator to correlate the derivedinformation with the associated particle downstream of the inspectionzone.

The collector may be of the same form as the focusing reflector asdescribed in accordance with previous aspects of the invention. In fact,the collector may also comprise part of the optical arrangement adaptedto direct electromagnetic radiation onto the flow of particles. In otherwords the collector may serve the dual function of collecting theproduced electromagnetic radiation as well as reflecting the incidentradiation onto the particles.

In accordance with another aspect of the present invention there isprovided an analyzation instrument including: a first reflector having apartial ellipsoidal shape; a near focal point of the partial ellipsoidalshape of the first reflector; a distant focal point of the partialellipsoidal shape of the first reflector; a central axis of the partialellipsoidal shape defined by the near focal point and distant focalpoint of the partial ellipsoidal shape of the first reflector; a sourceof electromagnetic radiation disposed at the near focal point of thepartial ellipsoidal shape capable of emitting electromagnetic radiationtoward the first reflector; a second reflector having a partialellipsoidal shape oriented relative to the first reflector so as to becapable of receiving electromagnetic radiation reflected by the firstreflector; a near focal point of the partial ellipsoidal shape of thesecond reflector; a distant focal point of the partial ellipsoidal shapeof the second reflector; a central axis of the partial ellipsoidal shapedefined by the near focal point and distant focal point of the partialellipsoidal shape of the second reflector; a flow source to produce aflow of particles to be analyzed; and an inspection zone of the flow ofparticles located at the near focal point of the partial ellipsoidalshape of the second reflector.

In a preferred embodiment, the source of electromagnetic radiation maycomprise an arc lamp. Further, a preferred relationship between thefirst reflector and the second reflector is that the distant focal pointof the first reflector and the distant focal point of the secondreflector overlap. The focal lengths of the first and second reflectorsmay be equivalent. Alternatively, the focal lengths of the tworeflectors may be different in that the first reflector has a greaterfocal length than the second reflector.

The term “ellipsoidal reflector” as used in the above described aspectof the invention and in following aspects and in the followingdescription of the invention, is understood to mean a reflector whichconforms to the shape of an ellipsoid of revolution. Furthermore, theterm is understood to mean a portion of a full ellipsoid of revolutionsuch as one third of an ellipsoid of revolution with an opening at thevertex.

In referring to ellipsoids throughout this description where only apartial ellipsoid is used, the near focal point is intended to mean thefocal point closest to the ellipsoidal portion being used.

In accordance with yet another aspect of the present invention there isprovided a method of analyzing including: utilizing a first reflectorhaving a partial ellipsoidal surface with a near focal point and adistant focal point; emitting electromagnetic radiation from a source ofelectromagnetic radiation positioned at the near focal point of thefirst reflector; reflecting electromagnetic radiation emitted by thesource of electromagnetic radiation from the first reflector; utilizinga second reflector having a partial ellipsoidal surface with a nearfocal point and a distant focal point; providing a flow of particles tobe analyzed; directing the flow of particles through an inspection zone;positioning the second reflector so that the near focal point of thesecond reflector overlaps the inspection zone and so that the secondreflector is capable of receiving electromagnetic radiation reflected bythe first reflector.

In accordance with another object of the present invention there isprovided an analyzation instrument including: a first reflector having apartial paraboloid shape; a focal point, and a focal length of thepartial paraboloid shape of the first reflector; a parabolic axis of thepartial paraboloid shape of the first reflector; a source ofelectromagnetic radiation disposed at the focal point of the partialparaboloid shape adapted to emit electromagnetic radiation toward thefirst reflector; a second reflector having a partial paraboloid shapeoriented relative to the first reflector so as to be capable ofreceiving electromagnetic radiation reflected by the first reflector; afocal point, and a focal length of the partial paraboloid shape of thesecond reflector; a parabolic axis of the partial paraboloid shape ofthe second reflector; a flow source to produce a flow of particles to beanalyzed; and an inspection zone of the flow of particles located at thefocal point of the partial paraboloid shape of the second reflector.

An arc lamp may be the source of electromagnetic radiation. It ispreferred that the parabolic axes, i.e., optical axes, of the first andsecond shapes-shapes are colinear. In one embodiment of the inventionthe focal lengths of the first and second reflectors may be equivalent.Alternatively the focal length of the first reflector may be greaterthan the focal length of the second reflector. A filter may be arrangedbetween the focal points of the two reflectors.

In another aspect of the present invention there is provided a method ofanalyzing including: utilizing a first reflector having a partialparaboloid surface, an optical axis and a focal point; emittingelectromagnetic radiation from a source of electromagnetic radiationpositioned at the focal point of the first reflector; reflectingelectromagnetic radiation emitted by the source of electromagneticradiation from the first reflector; utilizing a second reflector havinga partial paraboloid surface, an optical axis and a focal point;providing a flow of particles to be analyzed; directing the flow ofparticles through an inspection zone; positioning the second reflectorso that the focal point of the second reflector overlaps the inspectionzone and so that the second reflector is capable of receivingelectromagnetic radiation reflected by the first reflector.

The present invention also provides, in accordance with another aspectof the invention, a nozzle including an opening for a flow of particlesto flow through; a reflector coupled to the nozzle and oriented toreflect electromagnetic radiation at the flow of particles.

The reflector may take on various forms such as an ellipsoidalreflective surface or a paraboloid reflective surface, the reflector andthe nozzle may even be integral. In a preferred embodiment of theinvention, the flow of particles passes through an inspection zone and asource of electromagnetic radiation is provided to illuminate theinspection zone. Where the reflective surface is of the kind having afocal point, then it is preferred that the focal point of the reflectivesurface overlaps the inspection zone.

In preferred forms of the invention, the reflective surface may comprisea metal shape embedded in the nozzle. Alternatively, the reflectivesurface may comprise a reflective coating applied to the nozzle.Suitably, the focal point of the reflective surface can be external tothe nozzle. The nozzle may be adapted to receive electromagneticradiation through the opening in the nozzle to illuminate the reflectoror through the nozzle material itself, e.g. via light transmissionthrough a glass nozzle.

In accordance with a further aspect of the invention there is provided amethod of illuminating a flow of particles, the method including:providing a nozzle having a reflector coupled to the nozzle and orientedto reflect electromagnetic radiation; supplying a flow of particles;directing the flow of particles through the nozzle; reflectingelectromagnetic radiation with the reflector toward the flow ofparticles.

Another aspect of the invention provides a flow cytometer including: aflow source to produce a flow of particles to be analyzed, the flowsource being adapted to direct the flow of particles through aninspection zone; an optical arrangement including a source ofelectromagnetic radiation, the optical arrangement adapted to directelectromagnetic radiation onto the flow of particles, at the inspectionzone; a partial ellipsoidal collector to collect electromagneticradiation either produced or deflected from the particles, the collectorhaving an internal reflective surface of partial ellipsoidal shape withtwo foci and an optical axis oriented along a line between the two foci;the flow source being oriented such that the flow of particles issubstantially aligned with the optical axis.

The preferred form of the flow cytometer may be a jet-in-air flowcytometer. Most preferably, the flow cytometer enables sorting throughthe use of electrostatic plates.

A corresponding aspect of the invention provides a method of flowcytometry including passing a flow of particles to be analyzed throughan inspection zone; providing a focusing reflector having one or morefoci; converging electromagnetic radiation onto the flow of particles atthe inspection zone by reflection from the focusing reflector andaligning the inspection zone with one of the one or more foci.

BRIEF DESCRIPTION OF DRAWINGS

Further aspects of the present invention will become apparent from thefollowing description which is given by way of example only and withreference to the accompanying drawings in which.

FIG. 1( a) is a cross-sectional view of one embodiment of an opticalapparatus capable of producing an annular beam of electromagneticradiation;

FIG. 1( b) is a section through the beam of FIG. 1;

FIG. 1( d) is a perspective view of one embodiment of a prism for use inthe optical apparatus of FIG. 1( a);

FIG. 1( e) is a perspective view of an alternative form of a prism foruse in the optical apparatus of FIG. 1 (a);

FIG. 1( f) is a perspective view of an alternative prism arrangement foruse in the optical apparatus of FIG. 1 (a);

FIG. 1( g) is a perspective view of an alternative prism arrangement foruse in the optical apparatus of FIG. 1 (a);

FIG. 2 is sectional view of a paraboloid reflector;

FIG. 3 shows various views though a reflector which includestransmitting and reflecting surfaces;

FIG. 4 is a cross-sectional view of a possible embodiment for areflector apparatus;

FIG. 5 is a cross-sectional view of a possible embodiment for a detectorapparatus;

FIG. 6 is a cross-sectional view of one preferred embodiment of a flowcytometer in accordance with an aspect of the present invention;

FIG. 7 is a cross-sectional view of a second embodiment of a flowcytometer in accordance with an aspect of the present invention;

FIG. 8 is a cross-sectional view of a third embodiment of a flowcytometer in accordance with an aspect of the present invention;

FIG. 9 is a cross-sectional view of a fourth embodiment of a flowcytometer in accordance with an aspect of the present invention;

FIG. 10 is a cross-sectional view of a fifth embodiment of a flowcytometer in accordance with an aspect of the present invention;

FIG. 11 is a cross-sectional view of a sixth embodiment of a flowcytometer in accordance with an aspect of the present invention;

FIG. 12 is a cross-sectional view of a reflector incorporated into aflow nozzle design according to an aspect of the present invention;

FIG. 13 is a cross-sectional view of a seventh embodiment of a flowcytometer in accordance with an aspect of the present invention;

FIG. 14 is a cross-sectional view of an eighth embodiment of a flowcytometer in accordance with an aspect of the present invention; and

FIG. 15 is a cross-sectional view of a ninth embodiment of a flowcytometer in accordance with an aspect of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As mentioned earlier, the present invention includes a variety ofaspects, which may be combined in different ways. The followingdescriptions are provided to list elements and describe some of theembodiments of the present invention. These elements are listed withinitial embodiments, however it should be understood that they may becombined in any manner and in any number to create additionalembodiments. The variously described examples and preferred embodimentsshould not be construed to limit the present invention to only theexplicitly described systems, techniques, and applications. Further,this description should be understood to support and encompassdescriptions and claims of all the various embodiments, systems,techniques, methods, devices, and applications with any number of thedisclosed elements, with each element alone, and also with any and allvarious permutations and combinations of all elements in this or anysubsequent application.

Some embodiments of the invention are discussed in “A New OpticalConfiguration for Flow Cytometric Sorting of Aspherical Cells”, Int.Soc. Optical Engr., Proc. Of Adv. Tech. Analytical Cytology, 1997, byJohn C. Sharpe, Peter N. Schaare and Rainer Kunnemeyer; “RadiallySymmetric Excitation and Collection Optics for Flow Cytometric Sortingof Aspherical Cells”, Cytometry 29:363-370 (1997) by John C. Sharpe,Peter N. Schaare, and Rainer Kunnemeyer; and “A New OpticalConfiguration for Flow Cytometric Sorting of Bovine Spermatozoa by Sex”,a thesis submitted to the University of Waikato for the degree of Doctorof Philosophy in Physics by Johnathan Charles Sharpe, which are herebyincorporated by reference.

FIG. 1( a) illustrates an optical apparatus including a prism 1. Theprism 1 has an apex 2 at a forward end of the prism, a right conicalportion having a conical face 2, and a right cylindrical base portioncontiguous with the conical portion. The base portion has a circularshaped rear end 4 with a reflective coating. An optical arrangement isprovided to provide incoming electromagnetic radiation 5 such asultra-violet light from a laser light source. The UV light 5 is directedin direction aligned with the central axis of the prism 1 onto the apex2 of the prism 1 via a second reflector in the form of mirror 6positioned at an angle of 45 degrees with respect to the incoming light5 and the central axis of the prism 1. As the incoming light 5 entersthe prism 1 via the apex 2 it is refracted by the prism 1 and divergesin a cone and is reflected off the rear end 4 of the lens 1. Thereflected light exits the prism 1 through its conical face 3 and isprojected from the forward end of the prism as an annular beam. The beamdefines an enclosed cylindrical band of light having a longitudinal axiscoincident with the central axis of the prism 1. FIG. 1( b) shows across section through the enclosed band of light. The production of acylindrical band of light may have many uses throughout the field ofoptics. FIG. 1( e) illustrates the prism 1 in perspective view.

FIG. 1 (d) illustrates an alternative form of prism 22. The prism 22 hasa right pyramidal portion with four inclined faces meeting at an apex. Abase portion is also provided which is square in cross-section,corresponding to the cross-section of the base of the pyramidal portion.The prism can be used in the same manner as prism 1 by directingincident light onto the apex of the prism in line with the central axisof the prism. However, in this embodiment, the projected light willemerge as four parallel beams equally spaced from the central axis. Thenumber of inclined faces of the pyramidal portion may vary, providedthat an even number is maintained.

FIG. 1 (f) illustrates an alternative prism arrangement in which areflective surface may be spaced from the rear end of the conical prismshown in FIG. 1 (e) or the pyramidal prism shown in FIG. 1 (d). Thespacing of the reflective surface 27 from the prism may be adjustable.

FIG. 1( g) illustrates an alternative prism arrangement known as aw-axicon or waxicon.

The waxicon 28 comprises an inner conical axicon surrounded by anannular axicon concentric with the inner axicon. The reflective surfacesdefine a W, hence the name waxicon.

FIG. 2 shows a paraboloid reflector 20 in the form of a minor having aparaboloidal-shaped internal reflective surface. The paraboloid internalreflective surface has a focus and an optical axis running through thefocus. It will be understood that the paraboloid shaped reflectivesurface can have the property whereby any light which leaves the focusof the paraboloid reflector and becomes incident on the surface of thereflector will be reflected out of the reflector 20 parallel to theoptical axis Likewise, when light which is reflected parallel to theoptical axis enters and hits the reflective surface, it will beprojected toward and through the focus. An aperture 21 is centrallypositioned within the paraboloid reflector 20, in line with the opticalaxis.

Thus, the paraboloid reflector 20 may be used to providemulti-directional illumination of an object for analysis or inspection.By positioning the object at the focus of the paraboloid reflector 20and providing light incident on the surface of the reflector 20 andparallel to the optical axis of the reflector 20, the incident light canbe reflected towards the object at the focus. Further, if the incomingparallel light is evenly spaced in relation to the optical axis then thelight illuminating the object at the focus will be radially symmetric.The paraboloid reflector 20 may thus be teamed with the opticalapparatus shown in FIG. 1 in a manner in which the paraboloid reflector20 is oriented to receive the light projected from the forward end ofthe prism 1 with the central axis of the prism 1 aligned with theoptical axis of the paraboloid reflector 20. This particular arrangementis discussed further in connection with the flow cytometer shown inFIGS. 6, 7,9,10,11,13. However the paraboloid reflector is not limitedin its use in combination with the optical apparatus shown in FIG. 1.

FIG. 3( a) (i) and (ii) are plan views of another embodiment of thesecond reflector of FIG. 1 generally indicated by arrow 30. The mirror30 includes reflective surfaces 31 and 32. The minor 30 also includes atransmitting portion which is in the form of an annular ring 33. Itshould be appreciated that in some embodiments the transmitting portion33 may be in the form of an aperture which extends through the minor 30.However, in other embodiments such as that shown more clearly in FIG. 3(b), the transmitting portion 33 may be in the form of a transparentmaterial, such as glass 34 which has not been covered by a reflectivesurface 35. As FIG. 3( b) shows, any incoming light 36 that impacts onthe reflective surface 35 is reflected, whereas incoming light whichimpacts on the transmitting portion 33 may continue to travelsubstantially in the same direction The transmitting portion 33 whenarranged at a 45 degree angle from which it is viewed in plan in FIG. 3(a) (i) serves to allow passage of the annular beam of light projectedfrom the forward end of the prism. FIG. 3( a) (ii) shows a plan view ofthe second reflector having an egg-shaped transmitting portion 33necessary to achieve the annular transmitting portion 33 when orientedat 45 degrees.

FIG. 4 shows an alternative reflector apparatus generally indicated byarrow 40 which may be used to collect illumination reflected from theparaboloid reflector 20 in FIG. 2. The reflector apparatus 40 includes abody 46 having a number of reflective surfaces 41, 42 and 43 which arepositioned with respect to the detector apparatus 40 so that they mayreflect any light they receive in different directions and/or atdifferent angles.

The reflector apparatus 40 also includes within its body 46 regions 44and 45 (both of which are bounded by dotted lines) which allow for thetransmission of light 47 through the reflector apparatus 40. It shouldbe appreciated that the regions 44 and 45 may be in the form ofapertures through the body 46 or alternatively made of a transparentsubstance/material capable of allowing for the transmission of light. Inembodiments where regions 44 and 45 are made of a transparentsubstance/material it will usually be desirable that the regions havethe same length as shown by double headed arrow x to ensure distancetraveled and refraction of the light 47 is substantially identical inboth regions.

The reflective surfaces 41, 42, and 43 are capable of discriminatingagainst the different types of light A, B and C that may be received bythe reflector apparatus 40, by reflecting it in different directionsand/or at different angles. Thus, the different types of light A, B andC may be reflected to suitable light detectors (not shown) fordetermination of the characteristics of each type of light.

FIG. 5 illustrates a detector apparatus generally indicated by arrow 50which may also be used to collect illumination from the paraboloidreflector shown in FIG. 2. In this embodiment the detector apparatus 50may also provide for the transmission of light 51 from a light source(not shown) in a similar manner to the reflector apparatus describedabove in connection with FIGS. 3 and 4. The detector apparatus 50 mayalso have a number of light detectors 52, 53 and 54 spatially positionedso that they may receive the different types of light A, B and Cincident on the reflector apparatus 50. Thus, the spatial orientation ofthe light detectors 52, 53 and 54 on the detector apparatus 50 allowsfor the discrimination between different types of light. On the otherhand, where measurement of certain light is not desired, eg. lightmerely reflected from the light source, such light can be allowed totravel through the transmitting portion(s) 51 of the detector apparatus.

FIG. 6 illustrates a first preferred embodiment of a flow cytometergenerally indicated by arrow 70. The flow cytometer 70 includes theoptical apparatus substantially as shown in FIG. 1. The opticalapparatus includes an optical arrangement including a light source 71and a minor 72. The light source 71 produces collimated ultra-violetlaser light 73 which is directed via mirror 72 to a prism 74 having acentral axis. The prism 74 is configured to produce a cylinder of light75 having a longitudinal axis coincident with the central axis of theprism. The prism may be the same as that indicated in FIG. 1 (a) or (e)of the drawings. Alternatively, the prism may have a pyramidal face suchas that shown in FIG. 1 (d) to produce parallel beams of light evenlyspaced from the central axis of the prism. The projected light 75 passesthrough an annular gap 76 in a second reflector 77 so as to be incidenton the 45 degree point of a paraboloid reflector/collector 78. It willbe seen in the following discussion that the reflector also services asa collector. For ease of reference the paraboloid reflector/collector 78will be simply referred to as the paraboloid reflector 78. Theparaboloid reflector 78 has an optical axis aligned with the centralaxis of the prism and a focus F lying on the optical axis.

Situated within the paraboloid reflector 78 is a nozzle assembly 79which delivers a particle stream 80 e.g. sperm cells, which issubstantially aligned with the optical axis of the paraboloid reflectorand passes through an inspection zone located at the focus F. The nozzleassembly 79 delivers the sperm cells in a saline sheath solution and mayutilize any of the known jet-in-air techniques to produce a laminar-flowparticle stream with the sperm flowing single file through theinspection zone at F.

The paraboloid reflector 78 is designed with two criteria in mind.Firstly, the reflector should be able to withstand the corrosiveenvironment introduced by the saline sheath environment. Secondly, thereflector should be designed to maximize reflectance of light of the UVfrequency. Either of a rhodium. reflective coating or an AlSiO₂reflective coating on a nickel substrate were found to be effective.

The effect of the cylinder of light 75 being incident at the 45 degreepoint of the paraboloid minor 78 is that it is reflected at 90 degreesso as to form a substantially coplanar disc of light which is convergenton the focal point F of the paraboloid reflector. Thus, this disc oflight is able to interact with the particle stream 80 and illuminate theparticles within the stream with substantially radially symmetricillumination.

If the particles have been stained with light-sensitive stain, theparticles will fluoresce when illuminated. The use of stains is anaccepted technique in sperm sexing since the number of molecules ofstain bound will be equivalent to the number of molecules of DNA. Thisdifference in uptake will yield a difference in the number of cellsavailable for excitation and fluorescence. The difference in DNA contentbetween X and Y sperm will yield a corresponding measurable differencein fluorescent light. Any of the known stains currently used for spermsexing may be used. In particular, Hoechst 33342 which is of thebis-benzimidazole family shown below has been shown to provide thenecessary X-Y differential resolution.

Thus, light which interacts with the particles will be scattered and/orfluoresced. This scattered and/or fluoresced light is then collected bythe paraboloid reflector/collector 78 and reflected parallel to theoptical axis of the paraboloid reflector 78. The second reflector 77 ispositioned at a substantially 45 degree angle so as to reflect thescattered and/or fluoresced light towards a light detector in the formof a photomultiplier tube 82. The second reflector 77 as appropriate maycomprise the forms illustrated in FIGS. 3-5.

For the specific application of the present invention in sexing sperm,the fluorescent light is of interest and the light merely scattered fromthe sperm in the sample stream may be of little or no interest. Thefluorescent light will be of a different frequency and the separation ofthe two frequencies can be achieved through the use of a high passfilter 200 positioned before the photo-multiplier tube 82.Alternatively, the separation of frequencies may be achieved through theuse of a dichroic mirror to reflect only those frequencies of interest.For example the dichroic minor may be incorporated into the secondreflector 77. However, if in certain applications it is desirable tomeasure scattered light, no filter is necessary.

It should be appreciated that instead of the single measurement detector82 shown, an array of measurement detectors may be provided with anappropriate array of filters for measuring different forms of light. Forexample, the use of a second reflector in the form as that shown in FIG.4 allows for the separation of light from different parts of theparaboloid reflector, it being possible to apply different filters toeach of the separate light parts.

Light which has not interacted with the particles may be refracted bythe medium which makes up the sample stream 80 and radiate as a disc inthe opposite direction to the incoming light. As the particle streamwill generally have a small diameter the resulting refraction of lightby the medium will not be great. Thus, this light will substantiallyretrace the path of the illuminating cylinder of light and exit throughthe annular gap 76 in the second reflector 77. This creates a simple yeteffective beam dump.

It should be appreciated that supporting structures of the components ofthe flow cytometer 70 including sample flow tubes for the nozzleassembly may obscure parts of the path for the cylinder of light 75.However, any resulting asymmetry in the disc of light is generallynegligible and the cylinder of light is therefore still consideredcylindrical. Optics might even be provided to refract an incident beamaround obstructions.

The amount of light measured by the photo-multiplier tube is passed to aprocessor, e.g., a computer (not shown) to derive predeterminedinformation such as an association between the amount of measured lightand a property of the cell from each of at least some of the particlesin the flow. This information is then correlated by a correlator, suchas a computer, with the corresponding particle downstream of theinspection zone to enable sorting of the particle depending whether itmeets certain selection criteria. For example, male and female sperm maybe sorted by sex.

The flow sorting technique uses electrostatics to charge and deflect acell containing droplet as it passes through an electric field. Thedroplet is created by a mechanical oscillation applied through apiezo-electric transducer thus perturbing the sample stream as it exitsthe nozzle 79. Each individual droplet can be charged depending on thecharacteristics of its contained particle just prior to break-off byapplication of a voltage to the carrier fluid. Depending on its charge,the droplet will be deflected from its normal gravitational trajectoryby oppositely charged plates 83. To incorporate droplet sorting it maybe necessary to provide a means by which to view the stream so as tocount the number of droplet spacings between the inspection point (i.e.the focal point F) and the break-off point of the droplets. This canusually be achieved by inserting a small periscope through the aperture84 in the base of the paraboloid reflector 78. Droplets which are notelectrostatically deflected from the central path are collected directlybelow and flushed to waste.

In FIG. 7 there is provided an alternative flow cytometer generallyindicated by arrow 100, this flow cytometer being substantially similarto the flow cytometer 70 shown in FIG. 6. Therefore, for ease ofreference, similar numbering has been used to illustrate the componentsused in this embodiment.

The major difference with this embodiment shown in FIG. 7 is that onlylight 101 collected from the upper regions of the paraboloid reflectorare received by the photomultipliers 102. Accordingly, the secondreflector 77 need not be of the type discussed in the previousembodiment. Instead, only a continuous minor confined within thecylindrical beam 75 need be used to reflect away the forward scatteredan r fluoresced light 103.

On the otherhand, it should also be readily appreciated that where it isonly desirable to consider forward scattered and/or fluoresced light,light measurement detectors may be suitable positioned so that they onlyreceive this light.

During experimentation, it was found that an increase in sample tosheath differential pressure resulted in increased positionaluncertainty of the particles through the focus, which results in adifference in illumination, and therefore fluorescence emission. Thereare a number of possible solutions which may be used either singly or incombination to broaden the focus around the sample stream.

The radial optics deliver a convergent disk of light at the excitationwavelength to the inspection point. Adjusting the vertical dimension ofthe radial focus is relatively simple if a concave or convex element ispositioned in the laser beam in front of the axicon. However, broadeningthe focus laterally, while retaining sufficient light intensity at thefocus for stain excitation and fluorescence, is not trivial.

To laterally broaden or defocus the radial focus requires that theillumination light cylinder be altered to cause divergence tangentiallyaround its circular cross-section. This would result in a lateraldisplacement of the incoming light disk thereby broadening the intensitydistribution of the focal area. Some optical elements were proposed toperform this function. The first optical element would take the form ofa radially etched diffraction grating. Such a component wouldsuccessfully achieve the goal of lateral displacement with a minimaldispersive effect in the vertical profile of the focus. The secondoptical element is a light shaping diffuser element. Implementation ofthis element into the radial optics design would result in both verticaland lateral focus broadening. Other options include a diffractor or acylindrical lens causing the beam to diffract sideways and broaden thefocus.

Another approach is to use the focusing characteristics of the laserbeam which is a Caussian beam where the depth of focus 1 is proportionalto the focal length f and inversely proportional to the beam diameter D.The variable L is defined as the half-height width of the flex densityprofile as plotted along the optical axis. Thus, an increase in thefocal length of the paraboloid reflector will cause an increase in d.Also, decreasing the diameter of the illuminating laser beam will bringabout an increase in d.

In another embodiment of the invention, paraboloid and ellipsoidalconfigurations of reflectors can be used to provide illumination of aninspection zone of a linear flow of particles. One distinct advantage ofthis type of system is the ability to use a low cost arc lamp to replacethe more expensive lasers commonly used in instruments of this type.Lasers are preferred in some devices because of the intensity of lightthat they can deliver. However, they have the disadvantage of onlyproviding a specific wavelength of electromagnetic radiation. Arc lamps,however, are less expensive and can provide many different wavelengthsof electromagnetic radiation in their emissions. Then, the properwavelength can be selected by use of an inexpensive filter which filtersout the undesired wavelengths of electromagnetic radiation.

Referring now to FIG. 8, an ellipsoidal embodiment of the invention canbe seen. FIG. 8 shows an analyzation instrument 201, such as a flowcytometer, in which a first reflector 200 having a partial ellipsoidalshape is disposed above a flow source which produces a flow 237 ofparticles to be analyzed. The reflector can be referred to as a partialellipsoidal reflector as it is essentially a halved ellipsoid.Nevertheless, it is understood that given the contour of its surface itis recognized as ellipsoidal or similarly having a partial ellipsoidalshape. This first reflector 200 has both a near focal point 202 disposednear the top of the ellipsoid shown in FIG. 8 and a distant focal point204 disposed below the partial ellipsoidal shape in FIG. 8. A centralaxis 208 of the partial ellipsoidal shape is defined by these two focalpoints.

A second reflector 216 can be disposed or oriented below the firstreflector. Again, the second reflector can have a partial ellipsoidalshape. Furthermore, the partial ellipsoidal shape can have a near focalpoint 220 disposed near the bottom of FIG. 8 and a distant focal point224 disposed overlapping or coincident with the distant focal point 204of the first reflector. In addition, the partial ellipsoidal shape ofthe second reflector can have a central axis 228 defined by its near anddistant focal points. Preferably, the central axis 208 of the firstreflector is substantially aligned with the central axis 228 of thesecond reflector.

A source of electromagnetic radiation, such as an arc lamp 212 can bedisposed at the near focal point of the first reflector 200. Due to theproperties of an ellipsoid, electromagnetic radiation emitted by thesource of electromagnetic radiation from the near focal point 202 andincident upon the first reflector 200 can be reflected back to thedistant focal point of the first reflector. When the distant focal point204 of the first reflector and the distant focal point 224 of the secondreflector are coincident and the central axis 208 of the first reflectorand the central axis 228 of the second reflector are collinear, thisreflected light can continue on a path such that it is incident upon thesecond reflector 216. The second reflector 216 can then reflect thelight which traveled through the distant focal point 224 of the secondreflector to the near focal point 220 of the second reflector. In thisfashion a real image of the source of electromagnetic radiation locatedat the near focal point 212 of the first reflector is created at thenear focal point 220 of the second reflector 216. Therefore, a veryintense light source can be concentrated on the inspection zone 236 ofthe linear flow of particles when the inspection zone is located at thenear focal point 220 of the second reflector. Furthermore, this allowsan arc lamp to be used—as a source with collimated beams, such as alaser, is unnecessary due to the ability of the reflectors to create areal image of the source of the electromagnetic radiation. Plus, afilter, such as a dichroic filter 240, can be used to filter out anywavelengths of undesired electromagnetic radiation.

When illuminated particles fluoresce, the fluorescence 215 can bereflected by the second reflector back towards a reflective surface,such as dichroic filter 240 which reflects the fluorescence to detectorhousing 244 to be detected. Because of the ellipsoidal geometry aconverging set of beams is created—thus, there is no need for optics tofocus the fluorescence on the detector. FIG. 8 also shows that a streamof cells can be deflected for sorting or analyzation purposes as theyfall through an opening in the second reflector 216.

In FIG. 8, the first reflector and second reflector are shown havingfocal lengths of f1 and f2 respectively. When these focal lengths areequivalent and the distant focal points are coincident and the centralaxes are aligned as shown, the real image of the arc lamp will be thesame size as the actual arc lamp. However, in some cases it is desirableto shrink the size of the real image of the arc source. This is the casewhen there is a possibility of two cells being very close to one anotherin the inspection zone of the stream. In such a case, it can beimportant to reduce a real image so that incident radiation is incidentupon only the cell under analyzation and not a second cell nearby. Thisprevents fluorescence from a second cell which might give an incorrectanalysis. There is more likelihood of cells being close by when thethroughput of the analyzer is increased.

The arrangement of FIG. 8 could be used with only the bottom reflectorand an alternative light source to illuminate the flow of particles.This might involve a laser directed at the flow of particles or off thereflective surface of the ellipsoidal reflector 216. This is a uniquearrangement in flow cytometry, because the flow of particles is alignedcoaxially with the central axis of the ellipsoidal reflector 216 to passthrough the near focal point of the ellipsoidal reflector 216. After theflow of particles passes through the focal point at which the particlesare irradiated with electromagnetic radiation for the purpose ofanalyzation, they can be sorted based upon their identifyingcharacteristics. Electrostatic plates can be provided and disposed belowthe opening in the ellipsoidal reflective surface to deflect theparticles as they pass close to or between the electrostatic plates.This embodiment is particularly unique in jet-in-air types of flowcytometers.

In FIG. 9 a similar arrangement to that shown in FIG. 8 can be seen, themajor difference being that paraboloid shapes are being used for thereflectors. A first reflector 200 having a partial paraboloid shape, afocal point (or focus) 302 is disposed to reflect electromagneticradiation from a source of electromagnetic radiation, such as arc lamp312. The source of electromagnetic radiation can be positioned at thefocus of the paraboloid such that all emissions originating from thefocus and incident on the partial paraboloid are reflected in collimatedbeams 313 toward a second reflector 316. The first reflector 300 and thesecond reflector 316 each have parabolic axes 308 and 338 respectively.These axes can be aligned such that a real image of the electromagneticsource appears at the focal point (or focus) 320 of the second reflector316. A flow source 332 can provide a flow of particles 337 that flowsthrough the focal point 320 of the second reflector 316. The portion ofthe flow of particles that flows through the focal point can be referredto as the inspection zone 336 upon which the electromagnetic radiationis focused so as to analyze a cell falling through the inspection zone.

When the incident electromagnetic radiation is incident upon a cell inthe inspection zone, the stained cell can be caused to fluoresce. Thisfluorescence 315 can then be reflected by the second reflector 316toward a reflector, such as dichroic minor 340, which directs thefluorescence toward an optical apparatus 345 that focuses thefluorescence on a detector 344.

Once again, selection of equivalent focal lengths for the firstreflector f1 and second reflector f2 will provide a real image of thearc lamp of the same size at the focal point of the second reflector.Similarly, choosing a focal length for the second reflector that issmaller than the focal length of the first reflector will result in asmaller image that will help prevent error when large throughput ofcells is desired and consequently cells are close together at theinspection zone.

In FIGS. 8 and 9, one can see that plates can be provided to sort cellsas they exit the ellipsoidal or paraboloid shapes.

In another embodiment of the invention, a nozzle 400 can be providedwith a reflector coupled to the nozzle itself. In fact, the reflectorcan even be integral to the nozzle. This presents a significantadvantage to the user of the analyzing apparatus as there is no need foralignment of the components since the coupling can accomplish that task.Referring to FIGS. 10, II, 12 and 13 one can see how various embodimentsof such a nozzle could be implemented. In FIG. 10, a paraboloid nozzleis shown. The nozzle can be manufactured of a material such as glassthat permits the transmission of electromagnetic radiation, such asvisible light. Incident beams of electromagnetic radiation from a sourceof electromagnetic radiation, such as a laser source 520 in FIG. 11 passthrough the nozzle body and are incident on a reflector 402. Thereflector 402 is coupled to the nozzle itself rather than existingseparate from the nozzle. An opening 404 can be provided in the nozzleto allow a flow of particles 408 to flow through. The reflector 402 canbe oriented to reflect the incident electromagnetic radiation at theflow of particles 408.

Two possible shapes which can be used for the reflective surface of thereflector are a paraboloid and an ellipsoid. In FIG. 10, a paraboloidreflective surface 412 is shown while in FIG. 11, an ellipsoidalreflective surface 512 is shown. As explained elsewhere, an inspectionzone 416 can overlap a focal point(s) of the reflective surface, such asfocal point 420 of the paraboloid of FIG. 10 to produce the desiredreflection patterns.

The nozzle can be used with a source of electromagnetic radiation, suchas a laser source 520 as shown in FIG. 11. However, it is alsoenvisioned that an arc lamp or other source could be used as well. Thesource of electromagnetic radiation emits beams 450 which can bedirected at the reflective surface. When the electromagnetic radiationis incident upon a cell under analysis, fluorescence is created as shownby beams 451.

To create the reflective surface, a variety of designs are possible.First, the nozzle body could be shaped in a paraboloid or ellipsoidalshape and then coated with a reflective material 428 applied to thenozzle surface. Additionally, a reflector, such as a metal reflector 424could be inserted or embedded in the nozzle body as shown in FIG. 12. Itmight even be possible to rely on refractive properties which causeinternal reflection or even total internal reflection.

In FIG. 13, an embodiment is shown in which the nozzle is shaped suchthat the focal point 420 of the reflective surface is external to thenozzle. External is intended to means outside of or away from the nozzleborder, In such an embodiment, electromagnetic radiation could bedirected at the focal point without needing to traverse through thenozzle body.

Alternative embodiments of the invention can be seen in FIGS. 14 and 15.In FIG. 14, the radial optics configuration for a flow cytometer 500 cancombine 360 degree radial illumination and radially symmetric collectionof fluorescence from particles or cells as they pass through theinspection point. A glass cone 516 and a paraboloid reflector 528 can beused. The optical beam of a laser 508 can be steered onto the point ofthe glass cone. The beam can then be refracted into a divergent cone oflight which is retro-reflected to produce a cylinder of laser lightwhich encircles and is antiparallel to the input beam. This lightcylinder can then be reflected by a 45 degree elliptical ring mirror 512and aligned parallel to the optical axis of the paraboloid reflector528. The angle of incidence of the cylindrical beam at the reflector is45 degrees, causing the beam to form a coplanar convergent diskperpendicular to and focused on the sample stream.

Stained cells can be carried by the sample stream through the radialexcitation focus and caused to fluoresce. Much of the fluorescence canbe collected by the paraboloid reflector and projected out in acollimated beam onto an aspheric condensing lens 504. The lens can focusthe fluorescent light to a spot which is imaged by a microscopeobjective 520 into a photomultiplier tube (PMT) 501 and filter housing.Optical alignment of specimens flowing through the focal region of theparaboloid reflector can be achieved by adjusting the flow cell positionto maximize fluorescent signals from calibration microspheres. Theparaboloid reflector can have a hole or opening in the base throughwhich the sample stream can exit and where a jet observation camera anddroplet sorting mechanism 532 can be situated.

In FIG. 15, a simplified version of the geometry of FIG. 14 is shown.The fluorescence collection elements can be retained to provide radiallysymmetric detection of cells as they pass through the inspection pointof the flow cytometer. Excitation of cells can be performed by steeringa laser beam 608 onto the paraboloid reflector 628 at an incidence anglethat results in beam delivery from one direction similar to standardflow cytometer illumination. This can be accomplished by reflecting thebeam off minor 612. Detection of cells can be performed by a paraboloidreflector and aspheric lens combination. A single PMT, for example witha 400LP filter, can be positioned to collect all of the light focused bythe aspheric lens. An additional neutral density filter (ND=1.3) canalso be used to prevent saturation of the detector even at low PMTamplifier voltages.

The embodiment in FIG. 15 is particularly useful as it does not requireas extensive an alignment of optics as is required in other embodiments.An ellipsoidal collector could also be used to deliver the laser lightreflected from an adjusted minor 612 and to reflect fluorescence to becollected at the PMT. The embodiments in FIG. 15 and are particularlyadvantageous because of the simplistic substantially coaxial alignmentof the reflector with the detector.

It should be appreciated that the embodiments described in thisdescription rely on physical arrangements that may not permit total orperfect collection, transmission, symmetry, reflection, alignment, etc.due to physical limitations of minors, optics and physical orientationof equipment. In view of these limits, such properties still may beconsidered at the very least as substantial.

The application also discloses the use of reflectors (20, 78, 216, 316,400) having internal reflective surfaces shaped as three-dimensionalfigures of revolution, for example paraboloid or ellipsoid. Thereflectors (20, 78, 216, 316) focus light incident onto the reflectorsat one or more foci (F, 220, 320, 420). The reflectors may be used incombination with the optical apparatus including the prisms (1, 22, 24,26, 28). The reflectors (20, 78, 216,316) may be used in flow cytometersfor focusing light at a sample stream (237, 337) passing through thefocus (F, 220, 320, 420) of the reflector (20, 78, 216, 316). Thecollection of scattered and/or fluorescent light from an illuminatedsample stream (237, 337) in a flow cytometer may be achieved with theuse of a collector shaped as a figure of revolution e.g. paraboloid orellipsoid.

Aspects of the present invention have been described by way of exampleonly and it should be appreciated that modifications and additions maybe made thereto without departing from the scope thereof.

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. It involvesboth optical techniques as well as devices to accomplish the appropriateoptical technique. In this application, the optical techniques aredisclosed as part of the results shown to be achieved by the variousdevices described and as steps which are inherent to utilization. Theyare simply the natural result of utilizing the devices as intended anddescribed. In addition, while some devices are disclosed, it should beunderstood that these not only accomplish certain methods but also canbe varied in a number of ways. Importantly, as to all of the foregoing,all of these facets should be understood to be encompassed by thisdisclosure.

The discussion included in this application is intended to serve as abasic description. The reader should be aware that the specificdiscussion may not explicitly describe all embodiments possible; manyalternatives are implicit. It also may not fully explain the genericnature of the invention and may not explicitly show how each feature orelement can actually be representative of a broader function or of agreat variety of alternative or equivalent elements. Again, these areimplicitly included in this disclosure. Where the invention is describedin device-oriented terminology, each element of the device implicitlyperforms a function. Apparatus claims may not only be included for thedevice described, but also method or process claims may be included toaddress the functions the invention and each element performs. Neitherthe description nor the terminology is intended to limit the scope ofthe claims that will be included in this or any subsequent patentapplication.

It should also be understood that a variety of changes may be madewithout departing from the essence of the invention. Such changes arealso implicitly included in the description. They still fall within thescope of this invention. A broad disclosure encompassing both theexplicit embodiment(s) shown, the great variety of implicit alternativeembodiments, and the broad methods or processes and the like areencompassed by this disclosure and may be relied upon when drafting theclaims for any subsequent patent application. It should be understoodthat such language changes and broader or more detailed claiming may beaccomplished at a later date (such as by any required deadline) or inthe event the applicant subsequently seeks a patent filing based on thisfiling. With this understanding, the reader should be aware that thisdisclosure is to be understood to support any subsequently filed patentapplication that may seek examination of as broad a base of claims asdeemed within the applicant's right and may be designed to yield apatent covering numerous aspects of the invention both independently andas an overall system.

Further, each of the various elements of the invention and claims mayalso be achieved in a variety of manners. Additionally, when used orimplied, an element is to be understood as encompassing individual aswell as plural structures that may or may not be physically connected.This disclosure should be understood to encompass each such variation,be it a variation of an embodiment of any apparatus embodiment, a methodor process embodiment, or even merely a variation of any element ofthese. Particularly, it should be understood that as the disclosurerelates to elements of the invention, the words for each element may beexpressed by equivalent apparatus terms or method terms—even if only thefunction or result is the same. Such equivalent, broader, or even moregeneric terms should be considered to be encompassed in the descriptionof each element or action. Such terms can be substituted where desiredto make explicit the implicitly broad coverage to which this inventionis entitled. As but one example, it should be understood that allactions may be expressed as a means for taking that action or as anelement which causes that action. Similarly, each physical elementdisclosed should be understood to encompass a disclosure of the actionwhich that physical element facilitates. Regarding this last aspect, asbut one example, the disclosure of a “reflector” should be understood toencompass disclosure of the act of “reflecting”—whether explicitlydiscussed or not—and, conversely, were there effectively disclosure ofthe act of “reflecting”, such a disclosure should be understood toencompass disclosure of a “reflector” and even a “means for reflecting”Such changes and alternative terms are to be understood to be explicitlyincluded in the description.

Any acts of law, statutes, regulations, or rules mentioned in thisapplication for patent; or patents, publications, or other referencesmentioned in this application for patent are hereby incorporated byreference. Any priority case(s) claimed by this application is herebyappended and hereby incorporated by reference. In addition, as to eachterm used it should be understood that unless its utilization in thisapplication is inconsistent with a broadly supporting interpretation,common dictionary definitions should be understood as incorporated foreach term and all definitions, alternative terms, and synonyms such ascontained in the Random House Webster's Unabridged Dictionary, secondedition are hereby incorporated by reference. Finally, all referenceslisted in the list of References To Be Incorporated By Reference InAccordance With The Provisional Patent Application or other informationstatement filed with the application are hereby appended and herebyincorporated by reference, however, as to each of the above, to theextent that such information or statements incorporated by referencemight be considered inconsistent with the patenting of this/theseinvention(s) such statements are expressly not to be considered as madeby the applicant(s).

Thus, the applicant(s) should be understood to have support to claim andmake a statement of invention to at least: i) each of the opticaldevices as herein disclosed and described, ii) the related methodsdisclosed and described, iii) similar, equivalent, and even implicitvariations of each of these devices and methods, iv) those alternativedesigns which accomplish each of the functions shown as are disclosedand described, v) those alternative designs and methods which accomplisheach of the functions shown as are implicit to accomplish that which isdisclosed and described, vi) each feature, component, and step shown asseparate and independent inventions, vii) the applications enhanced bythe various systems or components disclosed, viii) the resultingproducts produced by such systems or components, ix) each system,method, and element shown or described as now applied to any specificfield or devices mentioned, x) methods and apparatuses substantially asdescribed hereinbefore and with reference to any of the accompanyingexamples, xi) the various combinations and permutations of each of theelements disclosed, xii) each potentially dependent claim or concept asa dependency on each and every one of the independent claims or conceptspresented, and xiii) all inventions described herein.

In addition and as to computer aspects and each aspect amenable toprogramming or other electronic automation, the applicant(s) should beunderstood to have support to claim and make a statement of invention toat least: xvi) processes performed with the aid of or on a computer asdescribed throughout the above discussion, xv) a programmable apparatusas described throughout the above discussion, xvi) a computer readablememory encoded with data to direct a computer comprising means orelements which function as described throughout the above discussion,xvii) a computer configured as herein disclosed and described, xviii)individual or combined subroutines and programs as herein disclosed anddescribed, xix) the related methods disclosed and described, xx)similar, equivalent, and even implicit variations of each of thesesystems and methods, xxi) those alternative designs which accomplisheach of the functions shown as are disclosed and described, xxii) thosealternative designs and methods which accomplish each of the functionsshown as are implicit to accomplish that which is disclosed anddescribed, xxiii) each feature, component, and step shown as separateand independent inventions, and xxiv) the various combinations andpermutations of each of the above.

With regard to claims whether now or later presented for examination, itshould be understood that for practical reasons and so as to avoid greatexpansion of the examination burden, the applicant may at any timepresent only initial claims or perhaps only initial claims with onlyinitial dependencies. Support should be understood to exist to thedegree required under new matter laws—including but not limited toEuropean Patent Convention Article 123(2) and United States Patent Law35 USC 132 or other such laws—to permit the addition of any of thevarious dependencies or other elements presented under one independentclaim or concept as dependencies or elements under any other independentclaim or concept. In drafting any claims at any time whether in thisapplication or in any subsequent application, it should also beunderstood that the applicant has intended to capture as full and broada scope of coverage as legally available. To the extent thatinsubstantial substitutes are made, to the extent that the applicant didnot in fact draft any claim so as to literally encompass any particularembodiment, and to the extent otherwise applicable, the applicant shouldnot be understood to have in any way intended to or actuallyrelinquished such coverage as the applicant simply may not have beenable to anticipate all eventualities; one skilled in the art, should notbe reasonably expected to have drafted a claim that would have literallyencompassed such alternative embodiments.

Further, if or when used, the use of the transitional phrase“comprising” is used to maintain the “open-end” claims herein, accordingto traditional claim interpretation. Thus, unless the context requiresotherwise, it should be understood that the term “comprise” orvariations such as “comprises” or “comprising”, are intended to implythe inclusion of a stated element or step or group of elements or stepsbut not the exclusion of any other element or step or group of elementsor steps. Such terms should be interpreted in their most expansive formso as to afford the applicant the broadest coverage legally permissible.

Finally, any claims set forth at any time are hereby incorporated byreference as part of this description of the invention, and theapplicant expressly reserves the right to use all of or a portion ofsuch incorporated content of such claims as additional description tosupport any of or all of the claims or any element or component thereof,and the applicant further expressly reserves the right to move anyportion of or all of the incorporated content of such claims or anyelement or component thereof from the description into the claims orvice-versa as necessary to define the matter for which protection issought by this application or by any subsequent continuation, division,or continuation-in-part application thereof, or to obtain any benefitof, reduction in fees pursuant to, or to comply with the patent laws,rules, or regulations of any country or treaty, and such contentincorporated by reference shall survive during the entire pendency ofthis application including any subsequent continuation, division, orcontinuation-in-part application thereof or any reissue or extensionthereon.

1-116. (canceled)
 117. An optical apparatus comprising: a flow source toproduce a flow of particles to be analyzed along a central axis, theflow source adapted to direct a flow of particles through an inspectionzone; a source of electromagnetic radiation for irradiating particles atthe inspection zone; an optical configuration, having an optical axis,to collect at least a portion of any electromagnetic radiation producedor deflected as a result of the illumination of the flow of particles,wherein the optical axis of the optical configuration is coaxial withthe central axis of the flow of particles; and a sensor for sensingradiation collected by the optical configuration.
 118. The opticalapparatus of claim 117 wherein the optical configuration comprises anobjective lens for focusing at least a portion of any electromagneticradiation produced or deflected as a result of the illumination of theflow of particles onto the sensor.
 119. The optical apparatus of claim117 wherein the optical configuration comprises a reflective surfacewith an optical axis which is coaxial with the central axis of the flowof particles.
 120. The optical apparatus of claim 117 wherein opticalconfiguration comprises a reflector with an optical axis which iscoaxial with the central axis of the flow of particles and an objectivelens.
 121. The optical apparatus of claim 120 wherein the optical axisof the objective lens is coaxial with the central axis of the flow ofparticles and an objective lens.
 122. The optical apparatus of claim 120further comprising a second reflector for reflecting light from thefirst reflector through the objective lens to the sensor.
 123. Theoptical apparatus of claim 117 wherein optical configuration comprises areflective surface with an optical axis or an objective lens with anoptical axis.
 124. The optical apparatus of claim 117 wherein theparticles comprises asymmetric particles.
 125. The optical apparatus ofclaim 124 wherein the asymmetric particles comprises sperm.
 126. Theoptical apparatus of claim 125 where the light collected by the opticalconfiguration is unaffected by sperm orientation.
 127. The opticalapparatus of claim 126 wherein the optical apparatus comprises a spermsorter without a sperm orienting feature.
 128. The optical apparatus ofclaim 125 wherein the sperm sorter does include an orienting feature.129. The optical apparatus of claim 120 wherein the reflector comprisesa reflector with one or more foci on an optical axis, wherein theoptical axis is coaxial with the central axis of the flow of particles.130. The optical apparatus of claim 117 wherein the optical apparatus isa flow cytometer.
 131. An optical apparatus comprising: a flow source toproduce a flow of particles to be analyzed along a central axis, theflow source adapted to direct a flow of particles through an inspectionzone; a source of electromagnetic radiation for irradiating particles atthe inspection zone; an objective lens, having an optical axis, forcollecting at least a portion of any electromagnetic radiation producedor deflected as a result of the illumination of the flow of particles,wherein the optical axis of the objective lens is coaxial with thecentral axis of the flow of particles; and a sensor for sensingradiation collected by the objective lens.
 132. The optical apparatus ofclaim 131 wherein the particles comprises asymmetric particles.
 133. Theoptical apparatus of claim 132 wherein the asymmetric particlescomprises sperm.
 134. The optical apparatus of claim 133 where the lightcollected by the optical configuration is unaffected by spermorientation.
 135. The optical apparatus of claim 134 wherein the opticalapparatus comprises a sperm sorter without a sperm orienting feature.136. The optical apparatus of claim 131 wherein the optical apparatus isa flow cytometer.
 137. An optical apparatus comprising: a flow path witha central axis for flowing particles to be analyzed through aninspection zone; a source of electromagnetic radiation for irradiatingparticles at the inspection zone; an optical configuration, having anoptical axis, to collect at least a portion of any electromagneticradiation produced or deflected as a result of the illumination of theflow of particles, wherein the optical axis of the optical configurationis coaxial with the central axis of the flow of particles; and a sensorfor sensing radiation collected by the optical configuration.
 138. Amethod of analyzing comprising the steps of: a. flowing particles to beanalyzed along a central axis through an inspection zone; b.illuminating the particles at the inspection zone with a source ofelectromagnetic radiation; c. collecting, with an optical configurationhaving an optical axis, at least a portion of any electromagneticradiation produced or deflected from the illumination of the flow ofparticles; and d. sensing a portion of the electromagnetic radiationproduced from the illumination of the flow of particles wherein theoptical axis of the optical configuration is substantially aligned withthe central axis along with the particles flow.
 139. The methodaccording to claim 138 wherein the substantially aligned optical axis iscoaxial with the central axis along which the particles flow.